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The Aurora-A kinase gene is frequently amplified and/or overexpressed in a variety of human cancers, leading to major efforts to develop therapeutic agents targeting this pathway. Here, we show that Aurora-A is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7 in a process that is regulated by GSK3β. Using a series of truncated Aurora-A proteins and site-directed mutagenesis, we identified distinct FBXW7 and GSK3β-binding sites in Aurora-A. Mutation of critical residues in either site substantially disrupts degradation of Aurora-A. Furthermore, we show that loss of Pten results in the stabilization of Aurora-A by attenuating FBXW7-dependent degradation of Aurora-A through the AKT/GSK3β pathway. Moreover, radiation-induced tumor latency is significantly shortened in Fbxw7+/−Pten+/− mice as compared with either Fbxw7+/− or Pten+/− mice, indicating that Fbxw7 and Pten appear to cooperate in suppressing tumorigenesis. Our results establish a novel posttranslational regulatory network in which the Pten and Fbxw7 pathways appear to converge on the regulation of Aurora-A level. Mol Cancer Res; 10(6); 834–44. ©2012 AACR.

Germ-line hypomorphism of the pleiotropic transcription factor Myc in mice, either through Myc gene haploinsufficiency or deletion of Myc enhancers, delays onset of various cancers while mice remain viable and exhibit only relatively mild pathologies. Using a genetically engineered mouse model in which Myc expression may be systemically and reversibly hypomorphed at will, we asked whether this resistance to tumour progression is also emplaced when Myc hypomorphism is acutely imposed in adult mice. Indeed, adult Myc hypomorphism profoundly blocked KRasG12D-driven lung and pancreatic cancers, arresting their evolution at the early transition from indolent pre-tumour to invasive cancer. We show that such arrest is due to the incapacity of hypomorphic levels of Myc to drive release of signals that instruct the microenvironmental remodelling necessary to support invasive cancer. The cancer protection afforded by long-term adult imposition of Myc hypomorphism is accompanied by only mild collateral side effects, principally in haematopoiesis, but even these are circumvented if Myc hypomorphism is imposed metronomically whereas potent cancer protection is retained.

The brain is actuated by billions of neurons with trillions of interconnections that regulate human behaviors. Understanding the mechanisms of these systems that induce sensory reactions and respond to disease remains one of the greatest challenges in science, engineering, and medicine. Recent advances in nanomaterials and nanotechnologies have led to the extensive research of electronic devices for brain interfaces to better understand the neural activities of the brain’s complex nervous system. The development of sensor devices for monitoring the physiological signals of the brain related to traumatic injury status has accompanied by the progress of electronic neural probes in parallel. In addition, these neurological and stereotactic surgical revolutions hold immense potential for clinical analysis of pharmacological systems within cerebral tissues. Here, we review the progress of electronic devices interfacing with brain in terms of the materials, fabrication technologies, and device designs. Neurophysiological activity can be measured and modulated by brain probes based on newly developed nanofabrication methodologies. Furthermore, in vivo pathological monitoring of the brain and pharmacological assessment has been developed in miniaturized and wireless form. We also consider the key challenges and prospects for further development, and explore the future directions emerging in the latest research.

Myc+/−haploinsufficient mice and mice with deletedMycenhancers express reduced levels of the pleiotropic transcription factor Myc. Such mice are viable, with relatively mild pathologies, but show delay in onset of certain cancers. However, many phenotypes arising from germline gene perturbation are indirect consequences of adaptive developmental compensation and so do not translate to equivalent phenotypes when applied to adults. To ascertain whether systemic Myc hypomorphism also conferred cancer protection when acutely imposed in adults, and what the side-effects of this might be, we constructed a genetically engineered mouse model in which Myc expression may be systemically and reversibly hypomorphed at will. Acute imposition of Myc hypomorphism in adult mice conferred potent protection against both KRasG12D-driven lung and pancreatic cancers yet elicited only mild haematopoietic side effects. These side effects were completely suppressed by imposing Myc hypomorphism metronomically – a regimen that nonetheless retained potent cancer prophylaxis. Our data identify a key bottleneck at the transition from pre-malignant hyperplasia to overt tumour that is peculiarly reliant on levels of Myc that are higher than those required for most adult physiology, offering a possible window of opportunity for Myc inhibition in cancer prophylaxis.

Interactions between proteins are fundamental for every biological process and especially important in cell signaling pathways. Biochemical techniques that evaluate these protein-protein interactions (PPIs), such as in vitro pull downs and co-immunoprecipitations, have become popular in most laboratories and are essential to identify and validate novel protein binding partners. Most PPIs occur through small domains or motifs, which are challenging and laborious to map by using standard biochemical approaches because they generally require the cloning of several truncation mutants. Moreover, these classical methodologies provide limited resolution of the interacting interface. Here, we describe the development of an alternative technique to overcome these limitations termed "Protein Domain mapping using Yeast 2 Hybrid-Next Generation Sequencing" (DoMY-Seq), which leverages both yeast-two-hybrid and next generation sequencing techniques. In brief, our approach involves creating a library of fragments derived from an open reading frame of interest and enriching for the interacting fragments using a yeast-two-hybrid reporter system. Next generation sequencing is then subsequently employed to read and map the sequence of the interacting fragment, yielding a high-resolution plot of the binding interface. We optimized DoMY-Seq by taking advantage of the well-described and high-affinity interaction between KRAS and CRAF, and we provide high resolution domain mapping on this and other protein interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2. Thus, DoMY-Seq provides an unbiased alternative method to rapidly identify the domains involved in PPIs by advancing the use of yeast two-hybrid technology.

Increased expression of ubiquitin-conjugating enzyme E2T (UBE2T) is reported in human prostate cancer. However, whether UBE2T plays any functional role in prostate cancer development remains unknown. We here report the first functional characterization of UBE2T in prostate carcinogenesis. Prostate cancer tissue array analysis confirmed upregulation of UBE2T in prostate cancer, especially these with distant metastasis. Moreover, higher level of UBE2T expression is associated with poorer prognosis of prostate cancer patients. Ectopic expression of UBE2T significantly promotes prostate cancer cell proliferation, motility and invasion, while UBE2T depletion by shRNA significantly inhibits these abilities of prostate cancer cells. Xenograft mouse model studies showed that overexpression of UBE2T promotes whereas UBE2T depletion inhibits tumor formation and metastasis significantly. Collectively, we identify critical roles of UBE2T in prostate cancer development and progression. These findings may serve as a framework for future investigations designed to more comprehensive determination of UBE2T as a potential therapeutic target.

A fully integrated low-power high-coexistence 2.4-GHz ZigBee transceiver implemented in 90-nm CMOS technology is demonstrated. The two-point direct-modulation with a fractional- N synthesizer is adopted in the transmitter architecture. The transmitter can provide high output power of +9 dBm and excellent error vector magnitude of 5.1%. The direct conversion is used in receiver for simplicity and -97-dBm minimum receiver sensitivity is achieved. Current consumptions for a TX at +9-dBm output power and for an RX are 28.4 and 15.4 mA, respectively. Excellent coexistence is presented through wireless local area network interferer rejection performance.

Endoplasmic reticulum (ER) stress has been implicated in Parkinson disease. We previously reported that thioredoxin 1 (Trx-1) suppressed the ER stress caused by 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine; however, its molecular mechanism remains largely unknown. In the present study, we showed that 1-methyl-4-phenylpyridinium ion (MPP(+)) induced ER stress by activating glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), tumor necrosis factor receptor-associated factor 2 (TRAF2), c-Jun N-terminal kinase (JNK), caspase-12, and C/EBP homologous protein (CHOP) in PC12 cells. The downregulation of Trx-1 aggravated the ER stress and further increased the expression of the above molecules induced by MPP(+). In contrast, overexpression of Trx-1 attenuated the ER stress and repressed the expression of the above molecules induced by MPP(+). More importantly, the overexpression of Trx-1 in transgenic mice suppressed ER stress by inhibiting the activation of these molecules. We present, for the first time, the molecular mechanism of Trx-1 suppression of endoplasmic reticulum stress in Parkinson disease in vitro and in vivo. Based on our findings, we conclude that Trx-1 plays a neuroprotective role in Parkinson disease by suppressing ER stress by regulating the activation of GRP78, IRE1α, TRAF2, JNK, caspase-12, and CHOP.

PMCID: PMC3388112.-- et al., The Aurora-A kinase gene is frequently amplified and/or overexpressed in a variety of human cancers, leading to major efforts to develop therapeutic agents targeting this pathway. Here, we show that Aurora-A is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7 in a process that is regulated by GSK3β. Using a series of truncated Aurora-A proteins and site-directed mutagenesis, we identified distinct FBXW7 and GSK3β-binding sites in Aurora-A. Mutation of critical residues in either site substantially disrupts degradation of Aurora-A. Furthermore, we show that loss of Pten results in the stabilization of Aurora-A by attenuating FBXW7-dependent degradation of Aurora-A through the AKT/GSK3β pathway. Moreover, radiation-induced tumor latency is significantly shortened in Fbxw7 +/-Pten +/- mice as compared with either Fbxw7 +/- or Pten +/- mice, indicating that Fbxw7 and Pten appear to cooperate in suppressing tumorigenesis. Our results establish a novel posttranslational regulatory network in which the Pten and Fbxw7 pathways appear to converge on the regulation of Aurora-A level. ©2012 AACR., J.-H. Mao was supported by the NIH, National Cancer Institute grant R01 CA116481, the Low Dose Scientific Focus Area, Office of Biological and Environmental Research, of the U.S. Department of Energy under contract no. DEAC02- 05CH11231, and Laboratory Directed Research and Development Program (LDRD). A. Balmain was supported by the Department of Energy Low Dose Radiation Research Program DESC0003679 and the National Cancer Institute grant U01 CA141455. J. Losada-Losada was partially supported by FEDER, MICINN (PLE2009-119), FIS (PI10/00328), and "Fundación Eugenio Rodríguez- Pascual."

The purpose of this study is to determine the correct estimation of laminate patterns for high-strength composites applied to a ship. Recently, the need for developing a ship component has been increasing to improve the capability of maritime operations. Composites with excellent specific strength and specific stiffness are emerging as next-generation materials. In the composite material, the mechanical properties vary depending on the laminated pattern of the reinforcing material. Therefore, in this study, the properties of the composite materials were calculated using the computer simulation program. The ply calibration performance results show that the initial values of the mechanical properties of the carbon/epoxy composites in the E11 direction are higher than the calculated values, and the remaining values are the same. The laminate mechanics results show that the tensile strength in the S11T direction was 1515 MPa, which is almost the same as the initial value of 1500 MPa.

Thioredoxin (TRX) is a key component of redox regulation and has been indicated to play an essential role in cell survival and growth. Here, we investigated the molecular mechanism of TRX in the regulation of cell survival and growth by using RNA interference (RNAi) in A549 lung cancer and MCF7 breast cancer cells. TRX knockdown did not significantly increase the basal level of cell death without exposure to stress, but CDDP-induced cell death was enhanced. Meanwhile, TRX knockdown resulted in significant cell-cycle arrest at the G(1) phase. Cyclin D1 expression was reduced by TRX knockdown at the protein and mRNA levels. TRX knockdown caused suppression of activation of the cyclin D1 promoter through elements including AP-1. TRX knockdown also reduced the levels of phosphorylated ERK1/2 and the nuclear translocation of ERK 1/2 induced by EGF. These results suggest that TRX is an important regulator of the cell cycle in the G(1) phase via cyclin D1 transcription and the ERK/AP-1 signaling pathways.

Exposure to excessive light induces retinal photoreceptor cell damage, leading to development and progression of various retinal diseases. We tested the effect of geranylgeranylacetone (GGA), an acyclic polyisoprenoid, on light-induced retinal damage in mice. Oral treatment with GGA (1.0 mg/d) for 5 d induced thioredoxin (Trx) and heat shock protein 72 (Hsp72) predominantly in the retinal pigment epithelium (RPE). After white light exposure (8000 lux for 2 h), the percentage of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive photoreceptor cells decreased significantly at 24 and 96 h, and the number of photoreceptor cell nuclei at 96 h and the electroretinographic amplitudes of the a- and b-waves at 4 and 10 d increased significantly in GGA-pretreated mice compared with saline-pretreated mice. Light-induced upregulations of 8-hydroxy-2-deoxyguanosine and 4-hydroxy-2-nonenal-modified protein, markers of oxidative stress, were inhibited by GGA pretreatment. To elucidate the cytoprotective mechanism of GGA and Trx, we used human K-1034 RPE cells and mouse photoreceptor-derived 661W cells. In K-1034 cells, GGA (10 μm) induced intracellular Trx, Hsp72, and extracellular Trx but not extracellular Hsp72. Extracellular Trx (0.75 nm) attenuated H2O2(200 μm)-induced cell damage in 661W cells. Pretreatment with GGA and overexpression of Trx in K-1034 cells counteracted H2O2(50 μm)-induced attenuation of cellular latex bead incorporation. Protection of phagocytotic activity through induction of Trx and possibly Hsp72 in RPE cells and elimination of oxidative stress in the photoreceptor layer through release of Trx from RPE cells may be mechanisms of GGA-mediated cytoprotection. Therefore, Trx is a neurotrophic factor released from RPE cells and plays a crucial role in maintaining photoreceptor cell integrity.

Background/Aims Thioredoxin is a small redox-active protein with anti-oxidant and anti-apoptotic effects. We have previously reported that thioacetamide-induced acute hepatitis was attenuated in thioredoxin transgenic mice. The aim of the present study was to investigate the protective effect of thioredoxin for hepatic fibrosis. Methods We subjected thioredoxin transgenic mice to thioacetamide-induced hepatic fibrosis. We also studied the effect of thioredoxin on the activation process of primary-cultured hepatic stellate cell. Results The expression of endogenous thioredoxin was induced in hepatocytes of thioacetamide-induced murine and rat fibrotic livers. Overexpression of thioredoxin inhibited tumor necrosis factor-α-induced apoptosis of HepG2 cells. Thioacetamide-induced fibrosis and accumulation of malondialdehyde were suppressed in transgenic mice as compared with wild type mice. Hepatic stellate cells isolated from transgenic mice were less proliferative than those isolated from wild type mice. Recombinant thioredoxin significantly inhibited DNA synthesis of primary-cultured stellate cells under serum or platelet-derived growth factor stimulation. Conclusions Thioredoxin has a potential to attenuate hepatic fibrosis via suppressing oxidative stress and inhibiting proliferation of stellate cells.

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4(+)-T cells derived from MIF -/- mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression.

Thioredoxin (TRX) is released from various types of mammalian cells despite no typical secretory signal sequence. We show here that a redox-active site in TRX is essential for its release from T lymphocytes in response to H2O2 and extracellular TRX regulates its own H2O2-induced release. Human T cell leukemia virus type I-transformed T lymphocytes constitutively release a large amount of TRX. The level of TRX release is augmented upon the addition of H2O2, but suppressed upon the addition of N-acetylcysteine. In the culture supernatant of a Jurkat transfectant expressing the tagged TRX-wild type (WT), the tagged TRX protein is rapidly released at 1 h and kept at a constant level until 6 h after the addition of H2O2. In contrast, another type of transfectant expressing the tagged TRX mutant (C32S/C35S; CS) fails to release the protein. H2O2-induced release of TRX from the transfectant is inhibited by the presence of rTRX-WT in a dose-dependent manner. Preincubation of the transfectant with rTRX-WT for 1 h at 37°C, but not 0°C, results in a significant suppression of the TRX release, reactive oxygen species, and caspase-3 activity induced by H2O2, respectively. Confocal microscopy and Western blot analysis show that extracellular rTRX-WT added to the culture does not obviously enter T lymphocytes until 24 h. These results collectively suggest that the oxidative stress-induced TRX release from T lymphocytes depends on a redox-sensitive event and may be regulated by negative feedback loops using reactive oxygen species-mediated signal transductions.

Thioredoxin (TRX) has a role in a variety of biological processes, including cytoprotection and the activation of transcription factors. Nerve growth factor (NGF) is a major survival factor of sympathetic neurons and promotes neurite outgrowth in rat pheochromocytoma PC12 cells. In this study, we showed that NGF induces TRX expression at protein and mRNA levels. NGF activated the TRX gene through a regulatory region positioned from -263 to -217 bp, containing the cAMP-responsive element (CRE). Insertion of a mutation in the CRE in this region abolished the response to NGF. NGF induced binding of CRE-binding protein to the CRE of the TRX promoter in an electrophoretic mobility shift assay. NGF also induced nuclear translocation of TRX. 2'-Amino-3'-methoxyflavone, an inhibitor of mitogen-activated protein kinase kinase, which is a known inhibitor of NGF-dependent differentiation in PC12 cells, suppressed the NGF-dependent expression and nuclear translocation of TRX. Overexpression of mutant TRX (32S/35S) or TRX antisense vector blocked the neurite outgrowth of PC12 cells by NGF. Overexpression of mutant TRX (C32S/C35S) suppressed the NGF-dependent activation of the CRE-mediated c-fos reporter gene. These results suggest that TRX plays a critical regulatory role in NGF-mediated signal transduction and outgrowth in PC12 cells.

Polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (MC) cause untoward effects including carcinogenesis. Here we investigated the effect of MC on apoptosis. MC induced apoptosis, preceded by serine 15 phosphorylation and accumulation of p53. MC failed to cause apoptosis in p53-deficient MG63 cells, whereas ectopic expression of p53 in MG63 cells restored the response to MC. Therefore, MC-induced apoptosis was dependent on p53. MC also activated p38 mitogen-activated protein kinase (MAPK) at 16-24 h. Accumulation of p53 and p53 phosphorylated at serine 15 was not changed by SB203580, a specific inhibitor of p38 MAPK or overexpression of a dominant negative mutant of p38 MAPK at 8 h after MC treatment, whereas the accumulation was suppressed at 24 h. These results suggest that MC induces accumulation and phosphorylation of p53 via a p38 MAPK-independent (early) and p38 MAPK-dependent (late) pathway. SB203580 repressed MC-induced apoptosis. MC induced p38 MAPK activation in p53 expressing cells but not in p53-deficient cells, indicating that the p38 MAPK activation was dependent on early p53 activation. The current study shows that both p53 and p38 MAPK activation are required for MC-induced apoptosis and provides a novel model of a functional regulation between p53 and p38 MAPK in chemical stress-induced apoptosis.

The ubiquitin ligase CUL4A has been implicated in tumorigenesis, but its contributions to progression and metastasis have not been evaluated. Here, we show that CUL4A is elevated in breast cancer as well as in ovarian, gastric, and colorectal tumors in which its expression level correlates positively with distant metastasis. CUL4A overexpression in normal or malignant human mammary epithelial cells increased their neoplastic properties in vitro and in vivo, markedly increasing epithelial–mesenchymal transition (EMT) and the metastatic capacity of malignant cells. In contrast, silencing CUL4A in aggressive breast cancer cells inhibited these processes. Mechanistically, we found that CUL4A modulated histone H3K4me3 at the promoter of the EMT regulatory gene ZEB1 in a manner associated with its transcription. ZEB1 silencing blocked CUL4A-driven proliferation, EMT, tumorigenesis, and metastasis. Furthermore, in human breast cancers, ZEB1 expression correlated positively with CUL4A expression and distant metastasis. Taken together, our findings reveal a pivotal role of CUL4A in regulating the metastatic behavior of breast cancer cells. Cancer Res; 74(2); 520–31. ©2013 AACR.

The ZigBee technology based on IEEE 802.15.4 is widespread use of biomedical applications providing wireless networks. A fully integrated low-power 2.4GHz ZigBee transceiver implemented in CMOS technology is demonstrated. It has RF and analog front-ends, a frequency synthesizer, and digital modulator and demodulator compliant to IEEE 802.15.4. The direct-modulation using fractional-N synthesizer is adopted as transmitter architecture. The transmitter provides high output power of +9dBm and excellent EVM of 5.1%. The direct conversion architecture uses in receiver. Receiver sensitivity is -97dBm. Current consumption for continuous TX transmission at +9dBm output power is 28.2mA and for continuous RX reception is 16mA. Excellence coexistence performance is presented by studying WLAN interferer rejection.

In a screen for thoracic malignancy-associated markers, thyroid stimulating hormone receptor (TSHR) was identified as a candidate as it binds to the previously-characterized lung cancer marker NKX2-1. We screened for mutations in all coding regions of the TSHR gene in 96 lung adenocarcinoma samples and their matched adjacent normal lung samples. We found one patient with a somatic mutation at codon 458 (exon 10), which is located at the transmembrane domain where most TSHR mutations have been found in thyroid-related diseases. This patient had lung adenocarcinoma with BAC (bronchioloalveolar carcinoma) features in the setting of a prior medical history significant for carotid stenosis and severe chronic obstructive pulmonary disease (COPD). In order to characterize the genetic features of TSHR in lung cancer, we checked for TSHR expression and copy number in the 96 lung cancer tissues. TSHR protein expression was generally overexpressed in multiple thoracic malignancies (adenocarcinoma, squamous cell carcinoma and malignant pleural mesothelioma) by immunohistochemistry. Our data suggest that aberrant TSHR function may contribute to lung cancer development or a subgroup of lung cancer with specific clinical phenotypes.

Mortality after initial diagnosis of lung cancer is higher than from any other cancer. Although mutations in several genes, such as EGFR and K-ras, have been associated with clinical outcome, technical complexity, cost and time have rendered routine screening prohibitive for most lung cancer patients prior to treatment. In this study, using both novel and established technologies, we developed a clinically practical assay to survey the status of three frequently mutated genes in lung cancer (EGFR, K-ras and TP53) and two genes (BRAF and β-catenin) with known hotspot mutations in many other cancers. A single 96-well plate was designed targeting a total of 14 fragments (16 exons) from EGFR, K-ras, TP53, BRAF and β-catenin. In 96 lung adenocarcinoma patients, the mutation frequencies of three major genes (EGFR, K-ras and TP53) were between 21-24%. Fifty-six out of 96 (58%) patients had a mutation in at least one of the five genes. K-ras mutations positively correlated with smoking pack-years (p=0.035). EGFR mutations were frequent in never-smokers (p=0.0007), Asians (p=0.0204) and non-stage I lung cancer (p=0.016). There was also a trend towards an association between the presence of any mutation and improved recurrence-free survival (p=0.070). We demonstrate that our novel multigene mutation assay technology can rapidly and cost-effectively screen for mutations in lung adenocarcinoma. This screening assay can be used in the clinical setting for the large-scale validation of prognosis and/or predicting therapeutic response so that the majority of lung cancer patients can benefit from leveraging up-to-date knowledge on how mutation profiles may influence treatment options.

The optimal deployment of multiple sensor nodes is an important issue in Wireless Sensor Networks (WSNs). Low power, network connectivity and coverage are major performance indicators in the deployment of sensor nodes. However, it is difficult to optimize all of the performance indicators due to trade-off among them. Satisfying user requirements for the deployment of sensor nodes is more important than optimizing the performance in an installation of sensor networks. This paper proposes a novel sensor-node deployment algorithm, which satisfies user requirements such as network lifetime, delay for the sensing data acquisition, and network coverage, using parameters of sensor nodes. Experiments also show that the proposed deployment satisfies the user requirements of sensor networks.

Indomethacin is one of non-steroidal anti-inflammatory drugs that are commonly used clinically and often cause gastric mucosal injury as a side effect. Generation of reactive oxygen species (ROS) and activation of apoptotic signaling are involved in the pathogenesis of indomethacin-induced gastric mucosal injury. Thioredoxin-1 (Trx-1) is a small redox-active protein with anti-oxidative activity and redox-regulating functions. The aim of this study was to investigate the protective effect of Trx-1 against indomethacin-induced gastric mucosal injury. Trx-1 transgenic mice displayed less gastric mucosal damage than wild type (WT) C57BL/6 mice after intraperitoneal administration of indomethacin. Administration of recombinant human Trx-1 (rhTrx-1) or transfection of the Trx-1 gene reduced indomethacin-induced cytotoxicity in rat gastric epithelial RGM-1 cells. Pretreatment with rhTrx-1 suppressed indomethacininduced ROS production and downregulation of phosphorylated Akt in RGM-1 cells. Survivin, a member of inhibitors of apoptosis proteins family, was downregulated by indomethacin, which was suppressed in Trx-1 transgenic mice or by administration of rhTrx-1 in RGM-1 cells. Trx-1 inhibits indomethacin-induced apoptotic signaling and gastric ulcer formation, suggesting that it may have a preventive and therapeutic potential against indomethacin-induced gastric injury.

Thioredoxin-1 (TRX) plays important roles in cellular signaling by controlling the redox state of cysteine residues in target proteins. TRX is released in response to oxidative stress and shows various biologic functions from the extracellular environment. However, the mechanism by which extracellular TRX transduces the signal into the cells remains unclear. Here we report that the cysteine modification at the active site of TRX promotes the internalization of TRX into the cells. TRX-C35S, in which the cysteine at residue 35 of the active site was replaced with serine, was internalized more effectively than wild-type TRX in human T-cell leukemia virus-transformed T cells. TRX-C35S bound rapidly to the cell surface and was internalized into the cells dependent on lipid rafts in the plasma membrane. This process was inhibited by wild-type TRX, reducing reagents such as dithiothreitol, and methyl-beta-cyclodextrin, which disrupts lipid rafts. Moreover, the internalized TRX-C35S binds to endogenous TRX, resulting in the generation of intracellular reactive oxygen species (ROS) and enhanced cis-diamine-dichloroplatinum (II) (CDDP)-induced apoptosis via a ROS-mediated pathway involving apoptosis signal-regulating kinase-1 (ASK-1) activation. These findings suggest that the cysteine at the active site of TRX plays a key role in the internalization and signal transduction of extracellular TRX into the cells.

We show that 1-methyl-4-phenylpyridinium ion (MPP(+)), an active metabolite of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), induces cytotoxicity via endoplasmic reticulum (ER)- and mitochondria-mediated pathways, and thioredoxin-1 (TRX-1), a redox-active protein, prevents MPTP-induced neurotoxicity. TRX-1 overexpression suppressed reactive oxygen species and the ATP decline caused by MPP(+) in HepG2 cells. MPP(+) activated caspase-12 in PC12 cells and induced cytotoxicity in HeLa-rho(0) cells lacking mitochondrial DNA, as well as in the parental HeLa-S3 cells. TRX-1-transgenic mice demonstrated significant resistance to caspase-12 activation and the apoptotic decrease of dopaminergic neurons after MPTP administration, compared with wild-type C57BL/6 mice.